Outbreaks of contagious ecthyma (CE) have been reported in vaccinated sheep and studies were undertaken to investigate the causes of these vaccination failures. The vaccination procedure was very effective in inducing a lesion at the site of vaccination, but a proportion of sheep (17 percent) were not fully protected when reinoculated with CE virus 4 weeks later. The size of the primary vaccination lesions, virus neutralizing antibody titers and virus-specific lymphocyte stImulation indices could not be used to predict the degree of protective immunity. Measurement of the neutralizing antibody and virus-specific lymphocyte transformation responses suggested that there was a minimal systemic immune response following CE virus inoculation. Higher levels of systemic immunity may be induced by parenteral administration of live CE vaccines compared to the current procedure of inducing a localized skin infection. Replication of CE virus in buffy coat cells in vitro suggested that the virus may replicate in macrophages and therefore parenteral administration of vaccines may be feasible. Occurrence of CE in vaccinated sheep raised questions about possible variation of antigenic types of CE virus. It was found that cross-neutralization and delayed-type hypersensitivity tests could not be used to classify the CE viral isolates. However, analysis of the structural polypeptides of CE virions revealed differences among the isolates in the position of distinct polypeptide bands in the molecular weight region of 37,000-44,000 daltons, allowing the isolates to be classified into four groups. The polypeptides which varied among the different groups were shown to be located in the surface component of the virion. Unilateral cross reactions detected in cross-neutralization tests were found to correlate with classification of the isolates based on the position of the distinct polypeptide bands. Cross-immunity tests were performed in lambs using two isolates which did not cross-react in the cross-neutralization tests and in which differences in the polypeptide profiles were detected. Reinoculation with virulent sheep-passaged CE viruses overcame the immunity of the lambs. By contrast, there was protection against the less virulent cell culture-passaged CE viruses with cross-protection between the two isolates. These results suggest that virulent CE viral isolates may be responsible for the occurrence of CE in vaccinated animals rather than differences in antigenicity. Two epidemiological aspects of CE infection of sheep were also studied. Latent CE infections were investigated by treating CE-inoculated sheep with corticosteroids. Treatment induced recrudescence of lesions at sites of previous CE virus inoculation, but virus could not be isolated from these lesions. Hence, the existence of latent infections could not be confirmed, but it is unlikely that latent infections are important for the initiation of CE disease outbreaks. Importance of colostral immunity was investigated with ewes vaccinated 6 months prior to parturition. This vaccination did not result in sufficient colostral immunity to protect lambs from subsequent exposure to CE virus, however, the severity of the CE lesions may have been reduced. / Ph. D.

Submitted by (edna.saturno@ufrpe.br) on 2016-11-03T13:19:37Z No. of bitstreams: 1 Rosana Leo de Santana.pdf: 1515110 bytes, checksum: 27fa86f8f333295d8921b44bc59f3dcb (MD5) / Made available in DSpace on 2016-11-03T13:19:37Z (GMT). No. of bitstreams: 1 Rosana Leo de Santana.pdf: 1515110 bytes, checksum: 27fa86f8f333295d8921b44bc59f3dcb (MD5) Previous issue date: 2012-02-13 / Contagious ecthyma is a severe and proliferative viral disease affecting ovine and caprine species, and eventually men, caused by the contagious ecthyma virus (ECV) of the Parapoxvirus genus. The disease is spread worldwide, including in Brazil, where in the state of Pernambuco, due to its endemicity, can be mistaken by other vesicular diseases such as the Foot and mouth Disease, which requires its diagnostic differentiation. Meanwhile, the control of the infection in endemic regions is limited due to the difficulties in replicating the virus in cell cultures, for the development of vaccines. The purpose of this work is to evaluate the behavior of ECV samples in primary cultures of fetal caprine cornea cells, and also to establish an efficient molecular diagnostic method for field samples, and to analyze the phylogenetic profile of the virus samples studied. Crust samples from twenty-two sheep and seven goats from the states of Pernambuco, Bahia, Sergipe and Paraiba presenting the clinical symptoms of EC were inoculated in cell monolayers, during seven consecutive passages at weekly intervals. During all passages, starting at 24 hours post-infection was observed a cytopathic effect (CE) characterized by cell rounding, cell fusion with the formation of small syncytia, cytoplasmic inclusion and vacuolization. The intensity of cell detachment from the layers ranged from 25% to 100% which varied according to the viral sample. We concluded that the primary cultures of the fetal caprine cornea cell appeared highly permissible to replication of ECV and the isolated samples of ECV seemed to adapt to the utilized culture with a slight variation among samples, and the identification by PCR showed to be an applicable method to the differential diagnosis of vesicular diseases, especially to support the actions of PNEFA, where goats and sheep are considered sentinel animals. The phylogenetic analysis showed that ORFV sequences from the northeastern of Brazil are highly correlated showing identical sequences up to 99%. In addition, they present more than 80% sequence identity with other isolates of ORFV worldwide. / Ectima contagioso (EC) ou orf é uma doença infecto-contagiosa da pele causada pelo vírus do ectima contagioso (ECV) ou ORFV, que acomete principalmente ovinos e caprinos, e ocasionalmente o homem. Protótipo do gênero Parapoxvírus da família Poxviridae, está amplamente disseminado em todo mundo, inclusive no Brasil, especialmente na Região Nordeste, onde a caprinovinocultura é amplamente praticada para a produção de pele, carne e leite. Em Pernambuco, a doença ocorre de forma endêmica e pode ser confundida com enfermidades vesiculares, como a Febre Aftosa (FA), havendo a necessidade de diferenciação, sobretudo como suporte às ações do Programa Nacional de Erradicação da Febre Aftosa (PNEFA). Uma grande limitação no controle da doença é a dificuldade de replicação do vírus em cultivo celular, visando sua manipulação biotecnológica para a produção de vacinas. O objetivo deste trabalho é isolar e avaliar o comportamento de amostras de ECV em cultura primária de células de córnea fetal caprina, além de estabelecer um diagnóstico molecular eficiente para amostras de campo e avaliar o perfil filogenético das amostras de campo estudadas. Amostras de crostas de 22 ovinos e de sete caprinos que apresentavam sinais clínicos de EC, originários dos Estados de Pernambuco, Bahia, Sergipe e Paraíba, foram inoculadas em monocamadas de células epiteliais de córnea de feto caprino, durante sete passagens consecutivas, a intervalos semanais. Para realização do diagnóstico molecular foi utilizado para amplificação de um fragmento de 235pb do gene do envelope B2L o par de primers, PPP-3 e PPP-4 denominados pan-parapoxvirus. Observou-se em todas as passagens, a partir de 24 horas pós-infecção, efeito citopático (ECP) caracterizado pelo arredondamento celular, fusão com formação de pequenos sincícios, vacuolização e corpúsculos de inclusão citoplasmática, com intensidade de 25% a 100% de desprendimento da camada celular, que variou de acordo com a amostra viral. Conclui-se que as culturas de células primárias de córnea fetal caprina mostraram-se altamente permissíveis à replicação do ECV e que as amostras virais isoladas mostraram-se adaptadas ao cultivo utilizado, com pequena variação entre as amostras virais, e que, a identificação viral através da PCR é um método aplicável ao diagnóstico diferencial de enfermidades vesiculares, sobretudo como suporte às ações do PNEFA, no qual caprinos e ovinos são considerados animais sentinelas. A análise filogenética mostrou que as seqüências de ORFV do Nordeste do Brasil estão altamente relacionadas entre si, com seqüências idênticas acima de 99%. Além de apresentarem mais de 80% de identidade com seqüências de outros isolados de ORFV em todo mundo.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The immunostimulatory properties of inactivated parapoxvirus ovis (iPPVO) have been investigated in different animal species and experimental settings. This study investigated the effects of administration of iPPVO on selected events of the innate response in mice. Neutrophil activation, phagocytic activity of macrophages, serum bactericidal activity, induction and antiviral activity of interferon type I (IFN - I) and expression of several classes of cytokines were assayed following intraperitoneal inoculation of Mus musculus with iPPVO (107 TCID50). Serum from iPPVO-treated animals showed IFN-I activity against murine encephalomyocarditis virus (EMCV) between 6 and 12 hours post infection (hpi), as shown by plaque reduction. A significant activation of neutrophils at 6hpi was observed by NBT reduction test in animals treated with the iPPVO. Peritoneal macrophages from mice treated with iPPVO demonstrated a significant increase (p<0.01) in phagocytic activity against Candida albicans both in vivo (between 12 and 96 hpi) and in vitro (24 and 72 hpi). iPPVO treated mice showed increased serum bactericidal activity against Escherichia coli (p<0.05) at two periods (24 and 72 hpi). A second study evaluated the expression of cytokines in response to inoculation of iPPVO. For this, spleens and serum samples were collected from mice treated with iPPVO at different intervals after inoculation and subjected to quantification of messenger RNA (mRNA) by real time PCR (qRT-PCR) and detection/quantification of serum cytokines by ELISA. Quantification of mRNA identified a significant and transient increase in the expression of various cytokines, with variable magnitude and kinetics. mRNA expression of proinflammatory cytokines (IL-1β, TNF-α and IL-8) peaked at 24 hpi (5.4 times increase) and 48 hpi (3 and 10 times, respectively). A 15-fold increase in expression of INF-γ and 6-fold for IL-12 was observed at 48 and 24 hpi, respectively. An increase in the expression of self-regulatory cytokines (Th2) cells, especially IL-10 and IL-4 was detected at later periods (72 and 96 hpi) with peaks of 4.7 and 4.9 fold, respectively. The determination of the concentration of serum cytokines by ELISA showed an increase in IL-1β, TNF-α, IL- 12, IFN-γ and IL-10 with kinetics similar to that observed by qPCR, especially for IL-1 and INF-γ. In summary, these results demonstrate that inoculation with iPPVO stimulates transiently a number events associated with cellular and humoral innate immune responses. If taken together, these effects would likely contribute for the enhanced resistance to certain pathogens observed in animals treated with iPPVO. / As propriedades imunoestimulatórias do Parapoxvirus ovis inativado (iPPVO) têm sido verificadas em diferentes espécies animais e condições experimentais. No presente trabalho foram investigados os efeitos da administração do iPPVO sobre eventos da resposta inata de camundongos. Ativação de neutrófilos, atividade fagocítica de macrófagos, atividade bactericida do soro, indução e atividade antiviral do interferon tipo I (INF-I) e expressão de várias classes de citocinas foram investigados em diferentes intervalos após inoculação de Mus musculus pela via intraperitonial com iPPVO (dose 107 TCID50). O soro de animais tratados com iPPVO apresentou atividade de INF-I frente ao vírus da encefalomiocardite murina (EMCV) entre 6 e 12 horas pós inoculação (hpi), como demonstrado pela redução significativa de formação de placas virais. Uma significativa ativação dos neutrófilos circulantes foi observada pela técnica de redução do NBT em animais tratados com o iPPVO às 6 hpi. Macrófagos peritoneais de camundongos tratados com iPPVO demonstraram um aumento significativo (p<0,01) na atividade fagocítica frente a Candida albicans tanto in vivo (entre 12 e 96 hpi) quanto in vitro (24 e 72hpi). Camundongos tratados com iPPVO apresentaram aumento na atividade bactericida do soro frente à Escherichia coli (p<0,05) em dois períodos avaliados (24 e 72 hpi). Um segundo estudo avaliou a expressão de citocinas em resposta à inoculação do iPPVO. Para isso, amostras de baço e soro foram coletados de camundongos tratados com iPPVO em diferentes intervalos após a inoculação e submetidas a quantificação de RNA mensageiro (RNAm) por PCR em tempo real (qRT-PCR) e detecção/quantificação de citocinas no soro por ELISA. A quantificação de RNAm permitiu detectar um aumento significativo e transitório da expressão de várias citocinas, com magnitude e cinética variáveis. A expressão de RNAm das citocinas pró-inflamatórias (IL-1β, TNF-α e IL-8) atingiu o pico às 24 hpi (aumento de 5,4 vezes), 48 hpi (3 e 10 vezes, respectivamente). Um aumento de 15 vezes na expressão gênica do INF-γ, e de 6 vezes para a IL-12 foi observado às 48 e 24 hpi, respectivamente. Um incremento na expressão das citocinas auto-regulatórias (Th2), principalmente IL-10 e IL-4, foi detectado em períodos mais tardios (72 e 96 hpi) com picos de 4,7 e 4,9 vezes, respectivamente. A determinação da concentração das citocinas séricas por ELISA revelou um aumento nos níveis de IL-1β, TNF- α, IL-12, INF-γ e IL-10, com uma cinética similar à observada pela técnica de qPCR, especialmente para IL-1β e INF-γ. Em resumo, esses resultados demonstram que o tratamento com iPPVO estimula de forma significativa e transitória uma série de eventos celulares e humorais ligados à resposta imune inata. Esses efeitos, se considerados em conjunto, provavelmente contribuem para o aumento da magnitude da resposta imunológica a certos patógenos observada em animais tratados com o iPPVO.